Defective DNA Mismatch repair (MMR) causes cancer. MMR suppresses tumors primarily through four mechanisms: (A) repair of base substitution point mutations, such as G/T mismatches (B) repair of frameshift and small insertion-deletion mutations (commonly called MSI), (C) impaired DNA damage and failure to apoptose, (D) suppression of promiscuous DNA recombination (commonly called homeologous recombination). Different E. coli MutL homologues (MLH and PMS genes) and MutS homologues (MSH) interact combinatorially to create multiple MMR complexes with different roles in tumor suppression. Briefly, mammalian MLH/PMS proteins heterodimerize to form three distinct complexes, MLH1/PMS1, MLH1/PMS2 and MLH1/MLH3. These complexes interact with MSH2/MSH6 and MSH2/MSH6 heterodimers. This research program focuses on the mechanistic roles of individual MLH/PMS genes and associated factors in suppression of MMR deficient GI tumor initiation and progression. This continuing application builds upon the last funding period's discoveries. In Aim1 we will extend our research theme into the roles of MLH/PMS genes in suppression of promiscuous DNA recombination. This aim will provide important insights into in a poorly characterized mechanism of GI tumor suppression. In Aim 2 we will characterize the roles of MLH1 functional domains in suppression of homeologous recombination and DNA damage response. This aim will provide mechanistic insights into MMR tumor suppression and has potential translational significance of chemotherapy regimen choice for MLH1 mutation carriers. In Aim 3 we will follow up in greater depth our discovery that TLE6-like gene amplification accelerates progression of MMR deficient GI tumors. This aim will provide important insights into the mechanisms of MMR deficient GI adenoma-adenocarcinoma progression in vivo. The overall goal is to understand more precisely the roles of individual MLH/PMS and associated genes in mechanisms of MMR deficient GI tumor initiation and progression. This work is significant because a more in depth mechanistic understanding of GI tumor initiation and progression is necessary if we are ever to develop better approaches to block GI tumor initiation and progression to adenocarcinoma. This study is significant because it will add greater mechanistic depth to our knowledge of these processes. Additionally, impaired DNA damage response in MMR deficient CRC patients causes them to have worse clinical outcome when treated with certain chemotherapy drugs. This study's goal to help define more precisely which MLH1 domains are critical for the DNA damage response has important implications for deciding which chemotherapy regimen patients carrying MLH1 missense mutations are given.